Method of epitope discovery

ABSTRACT

A method of epitope discovery comprising the step of selecting an epitope from a population of peptide fragments of an antigen associated with a target cell, wherein the fragments have a known or predicted affinity for a major histocompatibility complex class I receptor peptide binding cleft, wherein the epitope selected corresponds to a proteasome cleavage product of the target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. patent application Ser. No. 09/561,074, filed onApr. 28, 2000, entitled “METHOD OF EPITOPE DISCOVERY;” which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention disclosed below relates to the identification of targetcell antigens that can be used to generate immunologically activecompositions. These compositions, when administered, will stimulate theimmune system of a subject to mount an immune response against a targetcell displaying the target antigen. The invention is contemplated tohave utility in the treatment and prevention of neoplastic and viraldisease.

2. Description of the Related Art

Neoplasia and the Immune System

The neoplastic disease state commonly known as cancer is thought togenerally result from a single cell growing out of control. Theuncontrolled growth state typically results from a multi-step process inwhich a series of cellular systems fail, resulting in the genesis of aneoplastic cell. The resulting neoplastic cell rapidly reproducesitself, forms one or more tumors, and eventually may cause the death ofthe host.

Because the progenitor of the neoplastic cell shares the host's geneticmaterial, neoplastic cells are largely exempt from the host's immunesystem. During immune surveillance, the process in which the host'simmune system surveys and localizes foreign materials, a neoplastic cellwill appear to the host's immune surveillance machinery as a “self”cell.

Viruses and the Immune System

In contrast to cancer cells, virus infection involves the expression ofclearly non-self antigens. As a result, many virus infections aresuccessfully dealt with by the immune system with minimal clinicalsequela. Moreover, it has been possible to develop effective vaccinesfor many of those infections that do cause serious disease. A variety ofvaccine approaches have been successfully used to combat variousdiseases. These approaches include subunit vaccines consisting ofindividual proteins produced through recombinant DNA technology.Notwithstanding these advances, the selection and effectiveadministration of minimal epitopes for use as viral vaccines hasremained problematic.

In addition to the difficulties involved in epitope selection stands theproblem of viruses that have evolved the capability of evading a host'simmune system. Many viruses, especially viruses that establishpersistent infections, such as members of the herpes and pox virusfamilies, produce immunomodulatory molecules that permit the virus toevade the host's immune system. The effects of these immunomodulatorymolecules on antigen presentation may be overcome by the targeting ofselect epitopes for administration as immunogenic compositions. Tobetter understand the interaction of neoplastic cells and virallyinfected cells with the host's immune system, a discussion of thesystem's components follows below.

The immune system functions to discriminate molecules endogenous to anorganism (“self” molecules) from material exogenous or foreign to theorganism (“non-self” molecules). The immune system has two types ofadaptive responses to foreign bodies based on the components thatmediate the response: a humoral response and a cell-mediated response.The humoral response is mediated by antibodies, while the cell-mediatedresponse involves cells classified as lymphocytes. Recent anticancer andantiviral strategies have focused on mobilizing the host immune systemas a means of anticancer or antiviral treatment or therapy.

The immune system functions in three phases to protect the host fromforeign bodies: the cognitive phase, the activation phase, and theeffector phase. In the cognitive phase, the immune system recognizes andsignals the presence of a foreign antigen or invader in the body. Theforeign antigen can be, for example, a cell surface marker from aneoplastic cell or a viral protein. Once the system is aware of aninvading body, antigen specific cells of the immune system proliferateand differentiate in response to the invader-triggered signals. The laststage is the effector stage in which the effector cells of the immunesystem respond to and neutralize the detected invader.

An array of effector cells implement an immune response to an invader.One type of effector cell, the B cell, generates antibodies targetedagainst foreign antigens encountered by the host. In combination withthe complement system, antibodies direct the destruction of cells ororganisms bearing the targeted antigen. Another type of effector cell isthe natural killer cell (NK cell), a type of lymphocyte having thecapacity to spontaneously recognize and destroy a variety of virusinfected cells as well as malignant cell types. The method used by NKcells to recognize target cells is poorly understood.

Another type of effector cell, the T cell, has members classified intothree subcategories, each playing a different role in the immuneresponse. Helper T cells secrete cytokines which stimulate theproliferation of other cells necessary for mounting an effective immuneresponse, while suppressor T cells down-regulate the immune response. Athird category of T cell, the cytotoxic T cell (CTL), is capable ofdirectly lysing a targeted cell presenting a foreign antigen on itssurface.

The Major Histocompatibility Complex and T Cell Target Recognition

T cells are antigen specific immune cells that function in response tospecific antigen signals. B lymphocytes and the antibodies they produceare also antigen specific entities. However, unlike B lymphocytes, Tcells do not respond to antigens in a free or soluble form. For a T cellto respond to an antigen, it requires the antigen to be bound to apresenting complex known as the major histocompatibility complex (MHC).

MHC complex proteins provide the means by which T cells differentiatenative or “self” cells from foreign cells. There are two types of MHC,class I MHC and class II MHC. T Helper cells (CD4⁺) predominatelyinteract with class II MHC proteins while cytolytic T cells (CD8⁺)predominately interact with class I MHC proteins. Both MHC complexes aretransmembrane proteins with a majority of their structure on theexternal surface of the cell. Additionally, both classes of MHC have apeptide binding cleft on their external portions. It is in this cleftthat small fragments of proteins, native or foreign, are bound andpresented to the extracellular environment.

Cells called antigen presenting cells (APCs) display antigens to T cellsusing the MHC complexes. For T cells to recognize an antigen, it must bepresented on the MHC complex for recognition. This requirement is calledMHC restriction and it is the mechanism by which T cells differentiate“self” from “non-self” cells. If an antigen is not displayed by arecognizable MHC complex, the T cell will not recognize and act on theantigen signal. T cells specific for the peptide bound to a recognizableMHC complex bind to these MHC-peptide complexes and proceed to the nextstages of the immune response.

As discussed above, neoplastic cells are largely ignored by the immunesystem. A great deal of effort is now being expended in an attempt toharness a host's immune system to aid in combating the presence ofneoplastic cells in a host. One such area of research involves theformulation of anticancer vaccines.

Anticancer Vaccines

Among the various weapons available to an oncologist in the battleagainst cancer is the immune system of the patient. Work has been donein various attempts to cause the immune system to combat cancer orneoplastic diseases. Unfortunately, the results to date have beenlargely disappointing. One area of particular interest involves thegeneration and use of anticancer vaccines.

To generate a vaccine or other immunogenic composition, it is necessaryto introduce to a subject an antigen or epitope against which an immuneresponse may be mounted. Although neoplastic cells are derived from andtherefore are substantially identical to normal cells on a geneticlevel, many neoplastic cells are known to present tumor-associatedantigens (TuAAs). In theory, these antigens could be used by a subject'simmune system to recognize these antigens and attack the neoplasticcells. Unfortunately, neoplastic cells appear to be ignored by thehost's immune system.

A number of different strategies have been developed in an attempt togenerate vaccines with activity against neoplastic cells. Thesestrategies include the use of tumor associated antigens as immunogens.For example, U.S. Pat. No. 5,993,828, describes a method for producingan immune response against a particular subunit of the Urinary TumorAssociated Antigen by administering to a subject an effective dose of acomposition comprising inactivated tumor cells having the Urinary TumorAssociated Antigen on the cell surface and at least one tumor associatedantigen selected from the group consisting of GM-2, GD-2, Fetal Antigenand Melanoma Associated Antigen. Accordingly, this patent describesusing whole, inactivated tumor cells as the immunogen in an anticancervaccine.

Another strategy used with anticancer vaccines involves administering acomposition containing isolated tumor antigens. In one approach, MAGE-A1antigenic peptides were used as an immunogen. (See Chaux, P., et al.,“Identification of Five MAGE-A1 Epitopes Recognized by Cytolytic TLymphocytes Obtained by In Vitro Stimulation with Dendritic CellsTransduced with MAGE-A1,” J. Immunol., 163(5):2928-2936 (1999)). Therehave been several therapeutic trials using MAGE-A1 peptides forvaccination, although the effectiveness of the vaccination regimes waslimited. The results of some of these trials are discussed in Vose, J.M., “Tumor Antigens Recognized by T Lymphocytes,” 10^(th) EuropeanCancer Conference, Day 2, Sep. 14, 1999.

In another example of tumor associated antigens used as vaccines,Scheinberg, et al. treated 12 chronic myelogenous leukemia (CML)patients already receiving interferon (IFN) or hydroxyurea with 5injections of class I-associated bcr-abl peptides with a helper peptideplus the adjuvant QS-21. Scheinberg, D. A., et al., “BCR-ABL BreakpointDerived Oncogene Fusion Peptide Vaccines Generate Specific ImmuneResponses in Patients with Chronic Myelogenous Leukemia (CML) [Abstract1665], American Society of Clinical Oncology 35^(th) Annual Meeting,Atlanta (1999). Proliferative and delayed type hypersensitivity (DTH) Tcell responses indicative of T-helper activity were elicited, but nocytolytic killer T cell activity was observed within the fresh bloodsamples.

Additional examples of attempts to identify TAAs for use as vaccines areseen in the recent work of Cebon, et al. and Scheibenbogen, et al. Cebonet al. Immunized patients with metastatic melanoma using intradermalllyadministered MART-1₂₆₋₃₅ peptide with IL-12 in increasing doses giveneither subcutaneously or intravenously. Of the first 15 patients, 1complete remission, 1 partial remission, and 1 mixed response werenoted. Immune assays for T cell generation included DTH, which was seenin patients with or without IL-12. Positive CTL assays were seen inpatients with evidence of clinical benefit, but not in patients withouttumor regression. Cebon, et al., “Phase I Studies of Immunization withMelan-A and IL-12 in HLA A2+ Positive Patients with Stage III and IVMalignant Melanoma,” [Abstract 1671], American Society of ClinicalOncology 35^(th) Annual Meeting, Atlanta (1999).

Scheibenbogen, et al. immunized 18 patients with 4 HLA class Irestricted tyrosinase peptides, 16 with metastatic melanoma and 2adjuvant patients. Scheibenbogen, et al., “Vaccination with Tyrosinasepeptides and GM-CSF in Metastatic Melanoma: a Phase II Trial,” [Abstract1680], American Society of Clinical Oncology 35^(th) Annual Meeting,Atlanta (1999). Increased CTL activity was observed in 4/15 patients, 2adjuvant patients, and 2 patients with evidence of tumor regression. Asin the trial by Cebon et al., patients with progressive disease did notshow boosted immunity. In spite of the various efforts expended to dateto generate efficacious anticancer vaccines, no such composition has yetbeen developed.

Vaccine strategies to protect against viral diseases have had manysuccesses. Perhaps the most notable of these is the progress that hasbeen made against the disease small pox, which has been driven toextinction. The success of the polio vaccine is of a similar magnitude.

Viral vaccines can be grouped into three classifications: liveattenuated virus vaccines, such as vaccinia for small pox, the Sabinpoliovirus vaccine, and measles mumps and rubella; whole killed orinactivated virus vaccines, such as the Salk poliovirus vaccine,hepatitis A virus vaccine and the typical influenza virus vaccines; andsubunit vaccines, such as hepatitis B. Due to their lack of a completeviral genome, subunit vaccines offer a greater degree of safety thanthose based on whole viruses.

The paradigm of a successful subunit vaccine is the recombinanthepatitis B vaccine based on the viruses envelope protein. Despite muchacademic interest in pushing the subunit concept beyond single proteinsto individual epitopes the efforts have yet to bear much fruit. Viralvaccine research has also concentrated on the induction of an antibodyresponse although cellular responses also occur. However, many of thesubunit formulations are particularly poor at generating a CTL response.

SUMMARY OF THE INVENTION

The invention disclosed herein is directed to the identification ofepitopes that are useful for generating vaccines capable of inducing animmune response from a subject to whom the compositions have beenadministered. One embodiment of the invention relates to a method ofepitope discovery comprising the step of selecting an epitope from apopulation of peptide fragments of an antigen associated with a targetcell, wherein the fragments have a known or predicted affinity for amajor histocompatibility complex class I receptor peptide binding cleft,wherein the epitope selected corresponds to a proteasome cleavageproduct of the target cell.

Another embodiment of the invention relates to a method of discoveringan epitope comprising the steps of: providing a sequence from a targetcell, wherein the sequence encodes or comprises a protein expressed inthe target cell; identifying a population of peptide fragments of theprotein, wherein members of the population of peptide fragments have aknown or predicted affinity for a major histocompatibility complex classI receptor peptide binding cleft; selecting the epitope from thepopulation of peptide fragments, wherein the epitope corresponds to aproduct of a proteasome active in the target cell.

One aspect of this embodiment relates an epitope discovered by theaforementioned method. Another aspect of this embodiment relates to avaccine comprising the discovered epitope. Still another aspect of theinvention relates to a method of treating an animal, comprisingadministering to the animal the aforementioned vaccine.

One embodiment of the disclosed invention relates to a method of epitopediscovery comprising the steps of: providing a neoplastic cell and asequence, wherein the sequence comprises or encodes an antigenassociated with the neoplastic cell; identifying a population of peptidefragments of the antigen, wherein the population of peptide fragments ispredicted to have an affinity for a major histocompatibility complexclass I receptor peptide binding cleft; selecting an epitope from thepopulation of peptide fragments, wherein the epitope is determine by invitro analysis to be a proteasome cleavage reaction product of aproteasome active in the neoplastic cell.

One aspect of this embodiment relates an epitope discovered by theaforementioned method. Another aspect of this embodiment relates to avaccine comprising the discovered epitope. Still another aspect of theinvention relates to a method of treating an animal, comprisingadministering to the animal the aforementioned vaccine.

Another embodiment of the disclosed invention relates to a method ofepitope discovery comprising the step of selecting an epitope from apopulation of peptide fragments of an antigen associated with a targetin a host, wherein the fragments have a known or predicted affinity fora major histocompatibility complex class II receptor peptide bindingcleft of the host, wherein the epitope selected corresponds to a productof proteolytic cleavage of the antigen in a cell of the host.

One aspect of this embodiment relates an epitope discovered by theaforementioned method. Another aspect of this embodiment relates to avaccine comprising the discovered epitope. Still another aspect of theinvention relates to a method of treating an animal, comprisingadministering to the animal the aforementioned vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically the parts of a cell involved in proteinprocessing by the proteasome and epitope presentation.

FIG. 2 depicts the results of a flow cytometry assay verifying HLAbinding by Melan-A epitopes.

FIG. 3 depicts the results of a flow cytometry assay verifying HLAbinding by Tyrosinase peptide 207-216.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention disclosed herein provide methods foridentifying epitopes of target antigens that can be used to generateimmunologically effective vaccines. Such vaccines can stimulate theimmune system to recognize and destroy target cells displaying theselected epitopes. Embodiments of the invention are particularly usefulin the treatment and prevention of cancers and of infections of cells byintracellular parasites, as well as in the treatment or prevention ofconditions associated with other pathogens, toxins, and allergens.

Certain kinds of targets are particularly elusive to the immune system.Among these are many kinds of cancer, as well as cells infected byintracellular parasites, such as, for example, viruses, bacteria, andprotozoans. A great deal of research has been done to identify usefulantigens and epitopes for generating an effective immune responseagainst such targets, with little success. This disclosure provides abasis for the efficient discovery of a new generation of effectiveepitopes effective against such elusive targets.

The invention disclosed herein makes it possible to select epitopesequences with true biological relevance. For an epitope to havebiological significance, e.g., to function in stimulating an immuneresponse, it must have an affinity for the binding cleft of a majorhistocompatibility complex (MHC) receptor peptide. There are variousmeans, known in the art, of predicting whether an oligopeptide sequencewill have an MHC binding affinity. However, most of the sequencespredicted to have MHC binding affinity are not biologically relevant,because they are not actually presented on the surface of a target cellor a pAPC.

The methods of the disclosed invention permit the vaccine designer toignore peptides that, despite predicted high binding affinity for MHC,will never be useful because they cannot be presented by target cells.Accordingly, methods and teachings disclosed herein provide a majoradvance in vaccine design, one that combines the power of antigensequence analysis with the fundamental realities of immunology. Themethods taught herein allows for the simple and effective selection ofmeaningful epitopes for creation of MHC class I or class II vaccinesusing any polypeptide sequence corresponding to a desired target.

Definitions

Unless otherwise clear from the context of the use of a term herein, thefollowing listed terms shall generally have the indicated meanings forpurposes of this description.

PROFESSIONAL ANTIGEN-PRESENTING CELL (PAPC)—a cell that possesses T cellcostimulatory molecules and is able to induce a T cell response. Wellcharacterized pAPCs include dendritic cells, B cells, and macrophages.

PERIPHERAL CELL—a cell that is not a pAPC.

HOUSEKEEPING PROTEASOME—a proteasome normally active in peripheralcells, and generally not present or not strongly active in pAPCs.

IMMUNE PROTEASOME—a proteasome normally active in pAPCs; the immuneproteasome is also active in some peripheral cells in infected tissues.

EPITOPE—a molecule or substance capable of stimulating an immuneresponse. In preferred embodiments, epitopes according to thisdefinition include but are not necessarily limited to a polypeptide anda nucleic acid encoding a polypeptide, wherein the polypeptide iscapable of stimulating an immune response. In other preferredembodiments, epitopes according to this definition include but are notnecessarily limited to peptides presented on the surface of cellsnon-covalently bound to the binding cleft of class I MHC, such that theycan interact with T cell receptors.

MHC EPITOPE—a polypeptide having a known or predicted binding affinityfor a mammalian class I or class II major histocompatibility complex(MHC) molecule.

HOUSEKEEPING EPITOPE—In a preferred embodiment, a housekeeping epitopeis defined as a polypeptide fragment that is an MHC epitope, and that isdisplayed on a cell in which housekeeping proteasomes are predominantlyactive. In another preferred embodiment, a housekeeping epitope isdefined as a polypeptide containing a housekeeping epitope according tothe foregoing definition, that is flanked by one to several additionalamino acids. In another preferred embodiment, a housekeeping epitope isdefined as a nucleic acid that encodes a housekeeping epitope accordingto the foregoing definitions.

IMMUNE EPITOPE—In a preferred embodiment, an immune epitope is definedas a polypeptide fragment that is an MHC epitope, and that is displayedon a cell in which immune proteasomes are predominantly active. Inanother preferred embodiment, an immune epitope is defined as apolypeptide containing an immune epitope according to the foregoingdefinition, that is flanked by one to several additional amino acids. Inanother preferred embodiment, an immune epitope is defined as apolypeptide including an epitope cluster sequence, having at least twopolypeptide sequences having a known or predicted affinity for a class IMHC. In yet another preferred embodiment, an immune epitope is definedas a nucleic acid that encodes an immune epitope according to any of theforegoing definitions.

TARGET CELL—a cell to be targeted by the vaccines and methods of theinvention. Examples of target cells according to this definition includebut are not necessarily limited to: a neoplastic cell and a cellharboring an intracellular parasite, such as, for example, a virus, abacterium, or a protozoan.

TARGET-ASSOCIATED ANTIGEN (TAA)—a protein or polypeptide present in atarget cell.

TUMOR-ASSOCIATED ANTIGENS (TuAA)—a TAA, wherein the target cell is aneoplastic cell.

HLA EPITOPE—a polypeptide having a known or predicted binding affinityfor a human class I or class II HLA complex molecule.

Note that the following discussion sets forth the inventor'sunderstanding of the operation of the invention. However, it is notintended that this discussion limit the patent to any particular theoryof operation not set forth in the claims.

Proteolytic Processing of Antigens

Epitopes that are displayed by MHC on target cells or on pAPCs arecleavage products of larger protein antigen precursors. For MHC Iepitopes, protein antigens are digested by proteasomes resident in thecell. Intracellular proteasomal digestion typically produces peptidefragments of about 3 to 23 amino acids in length. Additional proteolyticactivities within the cell, or in the extracellular milieu, can trim andprocess these fragments further. Processing of MHC II epitopes occursvia intracellular proteases from the lysosomal/endosomal compartment.See FIG. 1.

Presumably, most products of protein processing by proteasomes or otherprotease activities have little or no affinity for the binding cleft ofa particular MHC receptor peptide. However, the processing products thatdo have such an affinity are likely to be presented at some level ofabundance, by MHC at the cell surface. Conversely, if a givenoligopeptide sequence does not emerge intact from the antigen processingactivities of the cell, it cannot be presented at the cell surface,regardless of the predicted affinity of the sequence for MHC.

Vaccine design that focuses entirely on MHC affinity is fundamentallyflawed. The mere fact that a peptide have MHC binding affinity does notensure that such a peptide will make for a functional immunogen. Toprovide an epitope capable of eliciting an effective immune responseagainst a TAA, the peptide must have MHC binding affinity and be theproduct of cellular peptide generating systems. The methods of thedisclosed invention utilize both MHC binding affinity analysis andantigen processing analysis protocols to identify new epitopes ofinterest.

Correlating Predicted or Known MHC Binding with Proteolytic Processingof Antigens

To identify epitopes potentially effective as immunogenic compounds,predictions of MHC binding alone are insufficient. Embodiments of theinvention combine an analysis of MHC binding with an analysis ofproteolytic processing to identify epitopes that have both of theessential properties of a useful epitope: MHC affinity and correctproteolytic processing. Peptides having both of these properties arestrong candidates for vaccines and immunotherapies. Peptides lackingeither of these properties are unlikely to have any significantopportunity to function as effective epitopes.

Embodiments of the invention are capable of identifying epitopes derivedfrom TAAs for use in vaccines. The target antigens can be derived fromneoplastic cells; cells infected with a virus or other intracellularparasite; cells infected with other pathogens such as bacteria, fungi,protozoans, or prions; or from other pathogenic agents such as toxins,venoms, allergens; and the like. In short, embodiments of the method canbe applied to virtually any protein sequence, to identify thereinepitopes capable of generation by proteolysis and capable of binding toMHC. Accordingly, the invention is not limited to any particular targetor medical condition, but instead encompasses discovery of biologicallyrelevant MHC epitopes from any useful source.

In a preferred embodiment, the TAA is characteristic of a neoplasticcell and is thus defined as a tumor-associated antigen (TuAA). PreferredTuAAs include: differentiation antigens such as MelanA (MART-1), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,GAGE-2, p15; overexpressed embryonic antigens generally; overexpressedoncogenes and mutated tumor-suppressor genes such as p53, Ras,HER-2/neu; unique tumor antigens resulting from chromosomaltranslocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR1 andviral antigens, such as Epstein Barr virus antigens (EBVA) and the humanpapillomavirus (HPV) antigens E6 and E7. Other antigens of interestinclude prostate specific antigen (PSA), prostate stem cell antigen(PSCA), MAAT-1, GP-100, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE,p185erbB-2, p185erbB-3, c-met, nm-23H1, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p15, and p16. Other targetantigens are also contemplated.

A variety of methods are available and well known in the art to identifyTuAAs. Examples of these techniques include differential hybridization,including the use of microarrays; subtractive hybridization cloning;differential display, either at the level of mRNA or protein expression;EST sequencing; and SAGE (sequential analysis of gene expression). Thesenucleic acid techniques have been reviewed by Carulli, J. P. et al J.Cellular Biochem Suppl. 30/31:286-296, 1998 (hereby incorporated byreference in its entirety). Differential display of proteins involves,for example, comparison of two-dimensional polyacrylamide gelelectrophoresis of cell lysates from tumor and normal tissue, locationof protein spots unique or overexpressed in the tumor, recovery of theprotein from the gel, and identification of the protein usingtraditional biochemical or mass spectrometry sequencing techniques. Anadditional technique for identification of TuAAs is the SEREX technique,discussed in Türeci, Ö., Sahin, U., and Pfreundschuh, M., “Serologicalanalysis of human tumor antigens: molecular definition andimplications”, Molecular Medicine Today, 3:342, 1997, and herebyincorporated by reference in its entirety. Use of these and othermethods provides one of skill in the art the techniques necessary toidentify useful antigens for generating housekeeping and immune class Iepitopes, as well as class II epitopes for a vaccines. However, it isnot necessary, in practicing the invention, to identify a novel TuAA orTAA. Rather, embodiments of the invention make it possible to identifyuseful epitopes from any relevant protein sequence, whether the sequenceis already known or novel.

Analysis of TAA Fragments for MHC Binding

In order to identify biologically relevant epitopes, fragments withinthe TAA with a known or predicted affinity for MHC are identified. Theamino acid sequence of a TAA can be analyzed by a number of differenttechniques with which to identify peptide fragments having a known orpredicted affinity for the MHC peptide binding cleft. In one embodimentof the invention, TAA fragments are analyzed for their predicted abilityto bind to the MHC peptide binding cleft using a computer algorithm.Each allele of MHC specifies a particular epitope binding domain. Thus,for any given MHC allele, the candidate peptides can be screened forpredicted affinity thereto.

Examples of suitable computer algorithms for this purpose include thatfound at the world wide web page of Hans-Georg Rammensee, JuttaBachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An InternetDatabase for MHC Ligands and Peptide Motifs (accessible by hypertexttransfer protocol (http://) at134.2.96.221/scripts/h1aserver.dll/EpPredict.htm). Results obtained fromthis method are discussed in Rammensee, et al., “MHC Ligands and PeptideMotifs,” Landes Bioscience Austin, Tex., 224-227, 1997, which is herebyincorporated by reference in its entirety. Another site of interest isBIMAS (accessible at hypertext transfer protocol (http://) atbimas.dcrt.nih.gov/molbio/hla_bind), which also contains a suitablealgorithm. The methods of this web site are discussed in Parker, et al.,“Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains,” J. Immunol.152:163-175, which is hereby incorporated by reference in its entirety.

Using the Parker, et al. derived algorithm with the methods of theinvention would select peptides using a number of possible retentiontimes (i.e. half times of dissociation) to indicate a binding sequence.In one embodiment, peptides with an infinite retention time would beselected. In another embodiment, peptides with a retention time of 25minutes or more would be selected to indicate a binding sequence. Instill another embodiment, a retention time of 15 minutes or more wouldbe selected to indicate a binding sequence. In still another embodiment,a retention time of 10 minutes or more would be selected to indicate abinding sequence. Retention times of 9, 8, 7, 6, 5, 4, 3, 2, and 1minute are also contemplated.

As an alternative to predictive algorithms, a number of standard invitro receptor binding affinity assays are available to identifypeptides having an affinity for a particular allele of MHC. Accordingly,by the method of this aspect of the invention, the initial population ofpeptide fragments can be narrowed to include only those peptides havingan actual or predicted affinity for the selected allele of MHC.

Initially, peptide candidates for this analysis can include everypossible sequence of about 6 to 24 contiguous amino acids from theentire protein sequence of the TAA. In a preferred embodiment, thesequences can be from about 7 to 20 amino acids in length. In a morepreferred embodiment, the sequences can be from about 8 to 15 aminoacids in length. For sequence analysis to identify fragments withpredicted affinity for MHC I, a most preferred embodiment analyzes allpossible sequences of 9 or 10 contiguous amino acid fragments of theTAA. Analysis of the MHC affinity of the fragments can be conducted invitro or via computer analysis of the fragments.

Selected common alleles of MHC I, and their approximate frequencies, arereported in the tables below. TABLE 1 Estimated gene frequencies ofHLA-A antigens CAU AFR ASI LAT NAT Antigen Gf^(a) SE^(b) Gf SE Gf SE GfSE Gf SE A1 15.1843 0.0489 5.7256 0.0771 4.4818 0.0846 7.4007 0.097812.0316 0.2533 A2 28.6535 0.0619 18.8849 0.1317 24.6352 0.1794 28.11980.1700 29.3408 0.3585 A3 13.3890 0.0463 8.4406 0.0925 2.6454 0.06558.0789 0.1019 11.0293 0.2437 A28 4.4652 0.0280 9.9269 0.0997 1.76570.0537 8.9446 0.1067 5.3856 0.1750 A36 0.0221 0.0020 1.8836 0.04480.0148 0.0049 0.1584 0.0148 0.1545 0.0303 A23 1.8287 0.0181 10.20860.1010 0.3256 0.0231 2.9269 0.0628 1.9903 0.1080 A24 9.3251 0.03952.9668 0.0560 22.0391 0.1722 13.2610 0.1271 12.6613 0.2590 A9 unsplit0.0809 0.0038 0.0367 0.0063 0.0858 0.0119 0.0537 0.0086 0.0356 0.0145 A9total 11.2347 0.0429 13.2121 0.1128 22.4505 0.1733 16.2416 0.138214.6872 0.2756 A25 2.1157 0.0195 0.4329 0.0216 0.0990 0.0128 1.19370.0404 1.4520 0.0924 A26 3.8795 0.0262 2.8284 0.0547 4.6628 0.08623.2612 0.0662 2.4292 0.1191 A34 0.1508 0.0052 3.5228 0.0610 1.35290.0470 0.4928 0.0260 0.3150 0.0432 A43 0.0018 0.0006 0.0334 0.00600.0231 0.0062 0.0055 0.0028 0.0059 0.0059 A66 0.0173 0.0018 0.22330.0155 0.0478 0.0089 0.0399 0.0074 0.0534 0.0178 A10 unsplit 0.07900.0038 0.0939 0.0101 0.1255 0.0144 0.0647 0.0094 0.0298 0.0133 A10 total6.2441 0.0328 7.1348 0.0850 6.3111 0.0993 5.0578 0.0816 4.2853 0.1565A29 3.5796 0.0252 3.2071 0.0582 1.1233 0.0429 4.5156 0.0774 3.43450.1410 A30 2.5067 0.0212 13.0969 0.1129 2.2025 0.0598 4.4873 0.07722.5314 0.1215 A31 2.7386 0.0221 1.6556 0.0420 3.6005 0.0761 4.83280.0800 6.0881 0.1855 A32 3.6956 0.0256 1.5384 0.0405 1.0331 0.04112.7064 0.0604 2.5521 0.1220 A33 1.2080 0.0148 6.5607 0.0822 9.27010.1191 2.6593 0.0599 1.0754 0.0796 A74 0.0277 0.0022 1.9949 0.04610.0561 0.0096 0.2027 0.0167 0.1068 0.0252 A19 unsplit 0.0567 0.00320.2057 0.0149 0.0990 0.0128 0.1211 0.0129 0.0475 0.0168 A19 total13.8129 0.0468 28.2593 0.1504 17.3846 0.1555 19.5252 0.1481 15.83580.2832 AX 0.8204 0.0297 4.9506 0.0963 2.9916 0.1177 1.6332 0.0878 1.84540.1925^(a)Gene frequency.^(b)Standard error.

TABLE 2 Estimated gene frequencies for HLA-B antigens CAU AFR ASI LATNAT Antigen Gf^(a) SE^(b) Gf SE Gf SE Gf SE Gf SE B7 12.1782 0.044510.5960 0.1024 4.2691 0.0827 6.4477 0.0918 10.9845 0.2432 B8 9.40770.0397 3.8315 0.0634 1.3322 0.0467 3.8225 0.0715  8.5789 0.2176 B132.3061 0.0203 0.8103 0.0295 4.9222 0.0886 1.2699 0.0416  1.7495 0.1013B14 4.3481 0.0277 3.0331 0.0566 0.5004 0.0287 5.4166 0.0846  2.98230.1316 B18 4.7980 0.0290 3.2057 0.0582 1.1246 0.0429 4.2349 0.0752 3.3422 0.1391 B27 4.3831 0.0278 1.2918 0.0372 2.2355 0.0603 2.37240.0567  5.1970 0.1721 B35 9.6614 0.0402 8.5172 0.0927 8.1203 0.112214.6516 0.1329 10.1198 0.2345 B37 1.4032 0.0159 0.5916 0.0252 1.23270.0449 0.7807 0.0327  0.9755 0.0759 B41 0.9211 0.0129 0.8183 0.02960.1303 0.0147 1.2818 0.0418  0.4766 0.0531 B42 0.0608 0.0033 5.69910.0768 0.0841 0.0118 0.5866 0.0284  0.2856 0.0411 B46 0.0099 0.00130.0151 0.0040 4.9292 0.0886 0.0234 0.0057  0.0238 0.0119 B47 0.20690.0061 0.1305 0.0119 0.0956 0.0126 0.1832 0.0159  0.2139 0.0356 B480.0865 0.0040 0.1316 0.0119 2.0276 0.0575 1.5915 0.0466  1.0267 0.0778B53 0.4620 0.0092 10.9529 0.1039 0.4315 0.0266 1.6982 0.0481  1.08040.0798 B59 0.0020 0.0006 0.0032 0.0019 0.4277 0.0265 0.0055 0.0028 0^(c) — B67 0.0040 0.0009 0.0086 0.0030 0.2276 0.0194 0.0055 0.0028 0.0059 0.0059 B70 0.3270 0.0077 7.3571 0.0866 0.8901 0.0382 1.92660.0512  0.6901 0.0639 B73 0.0108 0.0014 0.0032 0.0019 0.0132 0.00470.0261 0.0060  0^(c) B51 5.4215 0.0307 2.5980 0.0525 7.4751 0.10806.8147 0.0943  6.9077 0.1968 B52 0.9658 0.0132 1.3712 0.0383 3.51210.0752 2.2447 0.0552  0.6960 0.0641 B5 unsplit 0.1565 0.0053 0.15220.0128 0.1288 0.0146 0.1546 0.0146  0.1307 0.0278 B5 total 6.5438 0.04354.1214 0.0747 11.1160 0.1504 9.2141 0.1324  7.7344 0.2784 B44 13.48380.0465 7.0137 0.0847 5.6807 0.0948 9.9253 0.1121 11.8024 0.2511 B450.5771 0.0102 4.8069 0.0708 0.1816 0.0173 1.8812 0.0506  0.7603 0.0670B12 unsplit 0.0788 0.0038 0.0280 0.0055 0.0049 0.0029 0.0193 0.0051 0.0654 0.0197 B12 total 14.1440 0.0474 11.8486 0.1072 5.8673 0.096311.8258 0.1210 12.6281 0.2584 B62 5.9117 0.0320 1.5267 0.0404 9.22490.1190 4.1825 0.0747  6.9421 0.1973 B63 0.4302 0.0088 1.8865 0.04480.4438 0.0270 0.8083 0.0333  0.3738 0.0471 B75 0.0104 0.0014 0.02260.0049 1.9673 0.0566 0.1101 0.0123  0.0356 0.0145 B76 0.0026 0.00070.0065 0.0026 0.0874 0.0120 0.0055 0.0028  0 — B77 0.0057 0.0010 0.01190.0036 0.0577 0.0098 0.0083 0.0034  0^(c) 0.0059 B15 unsplit 0.13050.0049 0.0691 0.0086 0.4301 0.0266 0.1820 0.0158  0.0059 0.0206 B15total 6.4910 0.0334 3.5232 0.0608 12.2112 0.1344 5.2967 0.0835  0.07150.2035  7.4290 B38 2.4413 0.0209 0.3323 0.0189 3.2818 0.0728 1.96520.0517  1.1017 0.0806 B39 1.9614 0.0188 1.2893 0.0371 2.0352 0.05766.3040 0.0909  4.5527 0.1615 B16 unsplit 0.0638 0.0034 0.0237 0.00510.0644 0.0103 0.1226 0.0130  0.0593 0.0188 B16 total 4.4667 0.02801.6453 0.0419 5.3814 0.0921 8.3917 0.1036  5.7137 0.1797 B57 3.59550.0252 5.6746 0.0766 2.5782 0.0647 2.1800 0.0544  2.7265 0.1260 B580.7152 0.0114 5.9546 0.0784 4.0189 0.0803 1.2481 0.0413  0.9398 0.0745B17 unsplit 0.2845 0.0072 0.3248 0.0187 0.3751 0.0248 0.1446 0.0141 0.2674 0.0398 B17 total 4.5952 0.0284 11.9540 0.1076 6.9722 0.10413.5727 0.0691  3.9338 0.1503 B49 1.6452 0.0172 2.6286 0.0528 0.24400.0200 2.3353 0.0562  1.5462 0.0953 B50 1.0580 0.0138 0.8636 0.03040.4421 0.0270 1.8883 0.0507  0.7862 0.0681 B21 unsplit 0.0702 0.00360.0270 0.0054 0.0132 0.0047 0.0771 0.0103  0.0356 0.0145 B21 total2.7733 0.0222 3.5192 0.0608 0.6993 0.0339 4.3007 0.0755  2.3680 0.1174B54 0.0124 0.0015 0.0183 0.0044 2.6873 0.0660 0.0289 0.0063  0.05340.0178 B55 1.9046 0.0185 0.4895 0.0229 2.2444 0.0604 0.9515 0.0361 1.4054 0.0909 B56 0.5527 0.0100 0.2686 0.0170 0.8260 0.0368 0.35960.0222  0.3387 0.0448 B22 unsplit 0.1682 0.0055 0.0496 0.0073 0.27300.0212 0.0372 0.0071  0.1246 0.0272 B22 total 2.0852 0.0217 0.82610.0297 6.0307 0.0971 1.3771 0.0433  1.9221 0.1060 B60 5.2222 0.03021.5299 0.0404 8.3254 0.1135 2.2538 0.0553  5.7218 0.1801 B61 1.19160.0147 0.4709 0.0225 6.2072 0.0989 4.6691 0.0788  2.6023 0.1231 B40unsplit 0.2696 0.0070 0.0388 0.0065 0.3205 0.0230 0.2473 0.0184  0.22710.0367 B40 total 6.6834 0.0338 2.0396 0.0465 14.8531 0.1462 7.17020.0963  8.5512 0.2168 BX 1.0922 0.0252 3.5258 0.0802 3.8749 0.09882.5266 0.0807  1.9867 0.1634^(a)Gene frequency.^(b)Standard error.^(c)The observed gene count was zero.

TABLE 3 Estimated gene frequencies of HLA-DR antigens CAU AFR ASI LATNAT Antigen Gf^(a) SE^(b) Gf SE Gf SE Gf SE Gf SE DR1 10.2279 0.04136.8200 0.0832 3.4628 0.0747 7.9859 0.1013 8.2512 0.2139 DR2 15.24080.0491 16.2373 0.1222 18.6162 0.1608 11.2389 0.1182 15.3932 0.2818 DR310.8708 0.0424 13.3080 0.1124 4.7223 0.0867 7.8998 0.1008 10.2549 0.2361DR4 16.7589 0.0511 5.7084 0.0765 15.4623 0.1490 20.5373 0.1520 19.82640.3123 DR6 14.3937 0.0479 18.6117 0.1291 13.4471 0.1404 17.0265 0.141114.8021 0.2772 DR7 13.2807 0.0463 10.1317 0.0997 6.9270 0.1040 10.67260.1155 10.4219 0.2378 DR8 2.8820 0.0227 6.2673 0.0800 6.5413 0.10139.7731 0.1110 6.0059 0.1844 DR9 1.0616 0.0139 2.9646 0.0559 9.75270.1218 1.0712 0.0383 2.8662 0.1291 DR10 1.4790 0.0163 2.0397 0.04652.2304 0.0602 1.8044 0.0495 1.0896 0.0801 DR11 9.3180 0.0396 10.61510.1018 4.7375 0.0869 7.0411 0.0955 5.3152 0.1740 DR12 1.9070 0.01854.1152 0.0655 10.1365 0.1239 1.7244 0.0484 2.0132 0.1086 DR5 unsplit1.2199 0.0149 2.2957 0.0493 1.4118 0.0480 1.8225 0.0498 1.6769 0.0992DR5 total 12.4449 0.0045 17.0260 0.1243 16.2858 0.1516 10.5880 0.11489.0052 0.2218 DRX 1.3598 0.0342 0.8853 0.0760 2.5521 0.1089 1.40230.0930 2.0834 0.2037^(a)Gene frequency.^(b)Standard error.

Tables 1, 2, and 3 derived from HLA Gene and Haplotype Frequencies inthe North American Population: The National Marrow Donor Program DonorRegistry, Mori, M. et al.

Determining Whether a Fragment with MHC Affinity is a Useful Epitope

As discussed above, a preliminary step of the disclosed method is toselect from among the original population of peptide fragments asubpopulation of peptides with an actual or predicted MHC affinity. Theselected fragments are analyzed further to determine which can beproduced by a cell under in vivo conditions that could result in bindingof the peptide to the selected MHC allele. All peptides that meet bothcriteria of MHC affinity and correct proteolytic processing aredesignated as “discovered epitopes.” A variety of methods are availablefor determining which peptide fragments can be produced by proteolyticprocessing in vivo. These methods include elution of peptides fromsolubilized MHC and intact cells, computer sequence analysis of theproteolytic cleavage motifs, and in vitro analysis of actual peptidefragments produced by cellular proteolytic machinery.

In a preferred embodiment, a series of synthetic peptides centrallycontaining either individual or clustered candidate peptide sequencescan be generated. Such peptides typically range in length from about 10to about 75 amino acids. In a preferred embodiment, the syntheticpeptide is between about 20 and 60 amino acids in length. In a morepreferred embodiment, the cluster is between about 30 and 40 amino acidsin length. Using standard peptide synthesis chemistry, including t-Bocprotection chemistry, Fmoc protection chemistry, and the like, one ofordinary skill in the art can produce a population of candidate peptidesfor subsequent screening.

Alternatively, peptide fragments containing candidate peptides can begenerated in vitro through protease digestion or chemical cleavage ofthe TAA or fragments thereof. Protease digestion to prepare suchfragments of TAAs can employ a wide variety of known proteases,including but not limited to proteasome proteases, trypsin,α-chymotrypsin, bromelain, clostripain, elastase, endoproteinases,exoproteinases, proteinase K, ficin, papain, pepsin, plasmin,thermolysin, thrombin, trypsin, cathepsins, and others. Chemical methodscan also be used to generate peptide candidates. Suitable chemicals orchemical reactions for cleaving peptide bonds include mild acidcleavage, cyanogen bromide, hydroxylamine, iodosobenzoic acid,2-Nitro-5-thiocyanobenzoate, and the like. In one embodiment, theunfragmented TAA can be used, although the use of a particularly largeinitial sequence can complicate the analysis.

Regardless of how the fragments containing candidate peptides arecreated, determining which epitopes are produced by the cellularmachinery is important. The immune and housekeeping proteasomes have thecapacity to cleave proteins at similar but distinct locations. Theimmune proteasome incorporates several subunits that distinguish it fromits housekeeping counterpart. These immune subunits include LMP2, LMP7,and MECL1, which replace the catalytic subunits of the housekeepingproteasome, and PA28α and PA28β, which serve a regulatory function.Collectively, incorporation of these subunits results in activity fromthe immune proteasome that is qualitatively and quantitatively differentfrom the activity of the housekeeping proteasome. In one embodiment ofthe invention, proteasome digestion is used to estimate cellular epitopegeneration. In this embodiment, immune and housekeeping proteasomes arepurified for in vitro use in order to assess the antigenic repertoiregenerated naturally from the two kinds of proteasomes. Differencesbetween immune proteasomes and housekeeping proteasomes, the epitopeproducts of these proteasomes, and the implications of these differencesfor vaccine design, are discussed in detail in copending U.S. patentapplication Ser. No. 09/560,465, filed on Apr. 28, 2000, entitledEPITOPE SYCHRONIZATION IN ANTIGEN PRESENTING CELLS, which isincorporated herein by reference in its entirety.

Epitopes presented by class I MHC on the surface of either pAPCs orperipheral cells are produced by digestion of proteins within thosecells by proteasomes. While it has been reported that the proteasomes ofpAPCs are not identical to the proteasomes of peripheral cells, thesignificance of this difference has been heretofore unappreciated. Thisinvention is based on the fact that when pAPCs and peripheral cellsprocess a given TAA, the proteasomes active in the pAPCs generateepitope fragments that are different from the epitope fragmentsgenerated by the proteasomes that are active in the peripheral cells.For convenience of reference, and as defined above, the proteasomes thatare predominantly active in pAPCs are referred to herein as “immuneproteasomes” while the proteasomes that are normally active inperipheral cells are referred to herein as “housekeeping proteasomes.”

The significance of the differential processing of TAAs by pAPCs andperipheral cells cannot be overstated. This differential processingprovides a unified explanation for why certain target cells areresistant to recognition and attack by the immune system. Although pAPCscan take-up TAAs shed from target cells or presented on their surface,the pAPCs will consequently stimulate the production of CTLs torecognize an “immune epitope” (the epitope resulting from processing ofthe TAA by the immune proteasome), whereas the target cells display“housekeeping epitopes” (distinct fragments of the TAA generated by thehousekeeping proteasome). As a consequence, the CTL response underphysiological conditions is misdirected away from epitopes on the targetcells.

Since CTL responses are induced by pAPCs, by definition they targetimmune epitopes rather than housekeeping epitopes and thus fail torecognize target cells, which are therefore able to persist in the body.This fundamental “epitope compartmentalization” of the cellular immuneresponse is the reason that some neoplastic cells can persist to formtumors; it is also the reason that some viruses and intracellularparasites can chronically infect cells without being eradicated by theimmune system. With regard to infectious agents, normally they cause theexpression of immune proteasomes in the cells they infect. This resultsin the production of epitopes on the cell surface that are identical tothose being presented by pAPCs to the immune system. Infection thusresults in “epitope synchronization” between the immune system and theinfected cell, subsequent destruction of the infected cells, andclearance of infectious agent from the body. In the case of someinfectious agents, notably those that are capable of establishingchronic infections, they have evolved a means of preventing expressionof immune proteasomes in the cells they infect. The proteasome in thesecells are maintained in a housekeeping mode, thereby preventing epitopesynchronization and attack by CTL. There is substantial evidence thatthis is a common mechanism used by virtually all chronic infectiousagents.

One way to overcome this failure on the part of CTLs to recognize anderadicate certain target cells is to provide vaccines and treatmentmethods that are capable of “synchronizing” epitope presentation.Epitope synchronization in this context means that the pAPCs are made topresent housekeeping epitopes, resulting in CTLs that can recognize thehousekeeping epitopes displayed on target cells, and thereby attack andeliminate the target cells.

Generally, proteasomes are prepared by affinity purification from cellextracts. In a preferred embodiment, a cell lysate is prepared usingstandard techniques. The lysate is cleared by ultracentrifugation iferythrocytes are not the original source material. The prepared celllysate is then purified from other cellular components using any one ofa number of purification techniques including various forms ofchromatography.

In one embodiment affinity chromatography is used to purify theproteasomes. The cell lysate is applied to an affinity column containinga monoclonal antibody (mAb) against one of the proteasomal subunits. Thecolumn is then washed to purify the bound proteasomes from othercellular material. Following washing, the bound proteasomes are theneluted from the column. The eluate is characterized in terms of proteincontent and proteolytic activity on a standard substrate.

Cleavage analysis using both housekeeping and immune proteasomes yieldsclass I epitopes from various TAA. The epitopes that are presented bypAPCs correspond to cleavage products of the immune proteasome, whilethe epitopes presented by tumors and by many cells chronically infectedwith intracellular parasites correspond to cleavage products of thehousekeeping proteasome. Once the digest is performed, the particularmolecular species produced are identified. In a preferred embodiment,this is accomplished by mass spectrometry. This allows the rapididentification of natural peptide fragments that are produced by eitherof the two kinds of proteasomes. In another embodiment, cleavage of thetarget antigen or fragments thereof by immune and housekeepingproteasomes, or by endosomal/lysosomal proteases (see below), ispredicted by computer modeling based on cleavage motifs of the relevantproteolytic activities.

Whereas class I MHC is loaded primarily with proteasomally derivedpeptides as it initially folds in the endoplasmic reticulum, the bindingcleft of class II MHC is blocked by the so-called invariant chain (Ii)in this compartment. Loading of peptide for class II MHC takes placeprimarily in the endosomal compartment, utilizing peptides generated byendosomal and lysosomal proteases. Thus if in vitro identification ofMHC class II epitopes is desired, preparations of proteases fromendosomal and/or lysosomal fractions can be substituted for theproteasomes. A variety of methods to accomplish this substitution aredescribed in the literature. For example, Kido & Ohshita, Anal.Biochem., 230:41-7 (1995); Yamada, et al., J. Biochem. (Tokyo),95:1155-60 (1984); Kawashima, et al., Kidney Int., 54:275-8 (1998);Nakabayshi & Ikezawa, Biochem. Int. 16:1119-25 (1988); Kanaseki &Ohkuma, J. Biochem. (Tokyo), 110:541-7 (1991); Wattiaux, et al., J. CellBiol., 78:349-68 (1978); Lisman, et al., Biochem. J. 178:79-87 (1979);Dean, B., Arch. Biochem. Biophys., 227:154-63 (1983); Overdijk, et al.,Adv. Exp. Med. Biol., 101:601-10 (1978); Stromhaug, et al., Biochem. J.,Biochem. J., 335:217-24 (1998); Escola, et al., J. Biol. Chem.271:27360-5 (1996); Hammond, et al., Am. J. Physiol., 267:F516-27(1994); Williams & Smith, Arch. Biochem. Biophys. 305:298-306 (1993);Marsh, M., Methods Cell Biol., 31:319-34 (1989); and Schmid & andMellman, Prog. Clin. Biol. Res., 270:35-49 (1988) all disclose methodsto prepare suitable proteolytic preparations. Each of the foregoingreferences is hereby incorporated by reference in its entirety.

In another embodiment, the digestion to determine which epitopes thecellular machinery produces, takes place within a cell expressing theTAA or a fragment thereof. For class I epitopes it is preferred that thetype of proteasome expressed by the cell be determined, for example, bywestern blotting. The MHC epitopes produced can then be eluted fromeither solubilized and purified MHC as described in Falk, K. et al.Nature 351:290, 1991, or directly from the intact cell as described inU.S. Pat. No. 5,989,565, both of which references are incorporatedherein by reference in their entirety. Eluted fragments are thenidentified by mass spectrometry.

Analysis of Target Protein Fragments

The molecular species detected by mass spectrometry are compared withthe candidate peptides predicted above. For the case of class Iepitopes, species that are as long as, or longer than, a candidatepeptide and share its C-terminus are desired; N-terminal trimming of atleast up to 25 amino acids can occur independently of the proteasome(Craiu, A. et al. Proc. Natl. Acad. Sci. USA 94:10850-55, 1997). ClassII MHC is much less limited in terms of the length of the peptides itwill bind, so the absence of cleavage in the middle of the epitopebecomes the primary criterion, rather than generation of a correct end.

A selected digestion product is then synthesized and used as a standardin an analytic method such as HPLC versus an aliquot of the digest. Thisprovides a further check on the identity of the digestion product andallows its yield to be determined. In rare cases more than one potentialproduct may have similar enough masses and chemical characteristics thatthey may not be reliably differentiated by these methods. In such casesthe HPLC peak can be collected and subjected to direct sequencing toconfirm identity.

Analysis of Peptides for MHC Binding

The epitope is synthesized and tested for its ability to bind a MHCreceptor. For example, in one preferred assay, cells displaying the MHCI receptor can be used to measure the binding affinity of candidatepeptides labeled with a radionuclide. Another preferred approachmeasures the ability of a peptide to bind to an MHC I receptor using acell culture-based assay. In this assay, cells lacking transportersassociated with antigen processing (TAP) are used to determine whetheror not a candidate peptide has the ability to bind to the MHC Ireceptor. TAP⁻ cells have the phenotype in which class I MHC proteins donot always fold properly, and surface expression of MHC I is thusreduced or abolished. When the cell is flooded with exogenous peptidethat can bind to the MHC I cleft, expression of the receptor isrestored. This can be monitored by several means such as RIA, FACS, andthe like. Using TAP⁻ cells, one of skill in the art can screen largenumbers of potential candidate peptides for receptor binding withouthaving to perform detailed binding affinity analysis.

The analysis methods of the various embodiments of the invention areuseful in examining candidate peptides generated in a variety of ways.For example, the described analysis can be used in evaluating multiplecandidate peptides generated through in vitro methods or bycomputational analysis, to identify those candidate sequences that haveMHC receptor binding characteristics. Preferred candidate peptides inthis embodiment of the invention are those that are already known to beproducts of proteolytic production by housekeeping and/or immuneproteasomes. Both in vivo cleavage products and in vitro cleavageproducts that are shown or predicted to bind to MHC are properlydesignated as “discovered epitopes.”

Epitope synchronization technology and vaccines for use in connectionwith this invention are disclosed in copending U.S. patent applicationSer. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION IN ANTIGENPRESENTING CELLS,” filed on Apr. 28, 2000, which is incorporated hereinby reference in its entirety (“As has been discussed herein, effectivecellular immunity is based on synchronized epitope presentation betweenthe pAPCs and the infected peripheral cells. In the absence of epitopesynchronization, target cells are not recognized by T cells, even ifthose T cells are directed against TAAs. Cancer cells and cellsharboring persistent intracellular parasites elude the cellular immuneresponse because they avoid epitope synchronization. “Natural” epitopesynchronization involves activation of immune proteasomes in infectedcells so that the infected cells display immune epitopes and are thusrecognized by T cells induced by pAPCs. Yet cancers and cells infectedby persistent intracellular parasites do not have active immuneproteasomes and thus go unrecognized by the normal array of induced Tcells. The vaccines and methods of preferred embodiments of the presentinvention thus represent, essentially, a “reverse” epitopesynchronization, causing the pAPCs to display housekeeping epitopes toaddress situations in which target cells do not display immune epitopes.. . . Certain embodiments also provide a second wave of epitopesynchronization by inducing pAPCs to display both housekeeping epitopesand immune epitopes corresponding to a selected target cell. Thus, inthese dual epitope embodiments, once the target cells are effectivelyattacked by T cells that recognize housekeeping epitopes, a switch bythe target cells to immune proteasome processing does not result in aloss of immune recognition. This is because of the presence of theimmune epitope in the vaccine, which acts to induce a population of Tcells that recognize immune epitopes. Preferred embodiments of thepresent invention are directed to vaccines and methods for causing apAPC or population of pAPCs to present housekeeping epitopes thatcorrespond to the epitopes displayed on a particular target cell. In oneembodiment, the housekeeping epitope is a TuAA epitope processed by thehousekeeping proteasome of a particular tumor type. In anotherembodiment, the housekeeping epitope is a virus-associated epitopeprocessed by the housekeeping proteasome of a cell infected with avirus. This facilitates a specific T cell response to the target cells.Concurrent expression by the pAPCs of multiple epitopes, correspondingto different induction states (pre- and post-attack), can drive a CTLresponse effective against target cells as they display eitherhousekeeping epitopes or immune epitopes.” “TILs isolated from patientbiopsies, or PBMCs from blood of donors or patients can be used toidentify housekeeping epitopes using methods that are commonly describedin the published literature. To identify housekeeping epitopes, thetarget cells used to test for active killing by PBMCs or TILs areconfirmed to express only the housekeeping proteasomes, and not toexpress at significant levels the immune proteasome. PBMCs from donorblood are stimulated in vitro using a panel of peptide antigens withpredicted affinity for the class I HLA allele expressed on the bloodcells being used. Each PBMC sample is stimulated with a specific class Ipeptide antigen for one week, preferably with the combination ofcytokines such as IL-2 or IL-12 to enhance the activity of the T cells.This stimulation is repeated at least three times to induce clonalexpansion of T cells specific against the peptide. A standard chromiumrelease assay is performed using target cells that are known to expressthe protein containing the epitope and exclusively the housekeepingproteasome. Evidence of killing of the target cells as measured bychromium release indicates that the peptide used to stimulate the PBMCsis present as a housekeeping epitope on the surface of the target cell.Tumors expressing this protein are thus candidate targets for a vaccinecontaining the epitope.”).

Epitope clusters for use in connection with this invention are disclosedin copending U.S. patent application Ser. No. 09/561,571 entitled“EPITOPE CLUSTERS,” filed on Apr. 28, 2000, which is incorporated hereinby reference in its entirety. Nucleic acid constructs useful as vaccinesin accordance with the present invention are disclosed in U.S. patentapplication Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODINGEPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2000, whichis incorporated herein by reference in its entirety.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES Example 1 Purification of Proteasome Complexes

A. Proteasome Complexes from Blood Cells

Concentrated erythrocyte bags were obtained from a local blood bank,(HemaCare, Van Nuys, Calif.). The contents of each bag were poured into200 ml centrifuge tubes and washed 3 times with PBS by centrifugation at2000 RPM for 10 minutes at room temperature in a swinging bucket rotorof a Megafuge 2.0 (Heraeus, Southplainfield, N.J.). After the last washthe samples were pooled in one container, to minimize variability amongtubes, and then re-divided into several centrifuge tubes. The cells werecentrifuged again at 2000 RPM for 10 min. The residual PBS wasaspirated. The pellet was stored at −70° C. until use.

B. Proteasome Complexes from Tumor Cells

Raji cells, a Burkitt's lymphoma cell line, were obtained from ATCC,(American Type Culture Collection, Manassas, Va.). The cells were grownusing standard cell culture methods and stimulated with INF-Gamma(100-500 U/ml) (Pharmingen, San Diego, Calif.). Expression of immuneproteasome subunits was confirmed separately by immunohistochemsitry onthe culture, and SDS-PAGE on a sample of the cell lysate. The cells werecollected by centrifugation, washed with PBS and stored at −70° C. untiluse.

C. Further Processing of Proteasome Complexes

Blood or lymphoma tumor cell pellets (frozen) were thawed in a 37° C.bath and ddH₂O was added to each tube. The cell suspension washomogenized in a 40 ml Dounce homogenizer. Further, for the tumor cells,the cell homogenate was centrifuged at 2000 rpm to remove cell debris.The supernatant was centrifuged at 10,000 rpm at 4° C. for 10 minutesand further centrifuged at 50,000 rpm at 4° C. for 30 minutes in aT-1270 rotor (Sorval, Newtown, Conn.).

The homogenates were passed through filter paper to remove debris, andthen pooled together. A 68% sucrose solution was added to the pooledhomogenate sample. An antibody-Sepharose preparation was incubated withthe homogenate for three hours at room temperature in a rotator. Thesuspension was centrifuged and washed 3× with TBS and further thoroughlywashed over vacuum funnel 6-8×. Proteasomes were eluted in TBS (pH 7.6)and the optical density of the eluate was measured. The proteasomepreparation was dialyzed overnight at 4° C. against 20 mM Tris (pH 7.6)using cellulose membrane MWCO 1000. The next day the proteasomepreparation was concentrated by ultrafiltration in a MilliporeULTRAFREE-15 centrifugation device (Millipore, Danbury, Conn.). Theproteasomes, at a concentration of 4 mg/ml, were then aliquotted andstored at −20° C. until use. The proteasomes were tested for activityand specificity by digestion of a fluorogenic substrate or a controlpeptide yielding known fragments. The following peptides are suitablefor use as control peptides for immune proteasome assays:MLLAVLYCLLWSFQTS (SEQ ID NO: 63); HSYTTAEEAAGITILTVILGVL (SEQ ID NO:64); EAASSSSTLVEVTLGEVPAAESPD (SEQ ID NO: 65); EFLWGPRALVETSYVKVLHHMVKI(SEQ ID NO: 66); APEEKIWEELSVLEVFEGR (SEQ ID NO: 67); andELMEVDPIGHLYIFAT (SEQ ID NO: 68). Underlined residues indicateproteolytic cleavage sites. Peptide FLWGPRALVETSYVK (SEQ ID NO: 69) issuitable as a control peptide for housekeeping proteasome assays.

D. Quantitation and Activity Analysis of Proteasome Preparations

An enzyme-linked immunosorbant assay (ELISA) was used to quantitate theproteasome preparations described above. ELISA techniques are well knownin the art and are discussed generally in Ausubel, et al., “ShortProtocols in Molecular Biology,” 3^(rd) Ed., Unit 11.2 (1997), which ishereby incorporated by reference in its entirety. Hybridoma cells(MCP-21) producing a monoclonal anti-human proteasome antibody wereobtained from the European Collection of Cell Culture ((ECACC), UK) andwere maintained using standard cell culture techniques and equipment.Hybridoma supplement (Gibco BRL, Rockville, Md.) was added to theantibody-producing cells. Upon reaching cell density of 500,000 cells/mlin an approximate volume of 2-3 liters, the cells were removed bycentrifugation and the supernatant was collected. Secretion of mAb inthe medium was monitored periodically by optical density (O.D.) using aLambda 20 Spectrophotometer (Perkin Elmer, Norwalk, Conn.).

The supernatant was passed over a protein G sepharose column(Amersham/Pharmacia Biotech Piscataway, N.J.). The column was washedwith PBS and the antibody was eluted in a 0.1M glycine buffer, pH 2.2.The optical density of the eluate fractions was measured at 280 nm, andthe positive factions were collected. The antibody was dialyzed against2 L of PBS for 2 days at 4° C. and stored until use.

The antibody was bound to CNBr-activated Sepharose 4B (AmershamPharmacia biotech, Piscataway, N.Y.). The antibody-Sepharose complex waswashed alternately 5-7 times with 0.1M sodium acetate saline, pH 4 and0.1M sodium borate saline, pH 8 and finally suspended in Tris bufferedsaline (TBS), pH 8. The preparation was stored at 4° C. until use.

E. Identification of Housekeeping and/or Immune Proteasomes by WesternBlotting

Both of the following protocols start with a membrane onto whichproteins extracted from cells of interest have been transferred afterelectrophoretic separation.

A. Chromogenic Protocol:

-   1. Wash the membrane for 5 min 20 ml PBS-T (phosphate buffered    saline, pH 7.4+0.1% Tween-20) at room temperature on an orbital    shaker (RT/shaker).    -   PBS (Sigma, Cat. No. P-3813)        -   (Volumes may vary with type of container throughout).-   2. Incubate the membrane for 5 min in 20 ml PBS-T, 3% H₂O₂ at    RT/shaker:    -   2 ml 30% H₂O₂+18 ml PBS-T-   3. Wash the membrane 3×5 min with PBS-T at RT/shaker.-   4. Block overnight in 20 ml PBS-T/5% nonfat dry milk at 4°    C./shaker:    -   20 ml PBS-T+1 g milk-   5. Rinse the membrane in PBS-T.

6. Incubate the membrane in 5 ml of primary antibody (Affinity ResearchProducts Ltd, United Kingdom) in blocking buffer for 2 hrs at RT/shaker:α-LMP 2 antiserum (mouse) (Cat. No. PW8205) 1:5000 α-LMP 2 antiserum(human) (Cat. No. PW 8345) 1:10000 α-LMP 7 antiserum (Cat. No. PW 8200)1:20000 α-20S proteasome α2 subunit monoclonal 1:1000 antibody (Cat. No.PW 8105)

These conditions are for the preceding antibodies only. Conditions forevery antibody must be determined empirically.

-   7. Wash the membrane as in step 3.-   8. Incubate the membrane in 5 ml of secondary antibody (Vector    Laboratories, Inc., Burlingame, Calif.) in blocking buffer for 30    min at RT/shaker:    -   GARB (Goat anti Rabbit) (for antisera) (Vector Labs Cat. No.        BA-1000) 1:2000    -   Horse anti mouse (for monoclonal antibodies) (Vector Labs Cat.        No. BA-2000) 1:1000-   9. Wash the membrane as in step 3.-   10. Incubate the membrane in 5 ml of ABC (Vector Laboratories, Cat.    No. PK-6100) in PBS-T for 30 min:    -   Make ABC at least 30 min before using as follows:    -   A=5 ul/1 ml=25 ul/5 ml    -   B=5 ul/1 ml=25 ul/5 ml    -   5 ul A+5 ul B>mix>let stand at 4° C.>add 990 ul PBS-T    -   Dilute ABC in PBS-T just before using-   11. Wash the membrane as in step 3.-   12. Detection:    -   1) transfer 5 ml of 0.2M PB into a 1^(st) 15 ml tube        -   0.4M Phosphate buffer:        -   90.4 ml of Sodium Phosphate Monobasic (1M)        -   619.2 ml of Sodium Phosphate Dibasic (0.5M)        -   pH to 7.4        -   QS to 1 L    -   2) transfer 2.8 ml of 0.2M PB into a 2^(nd) 15 ml tube    -   3) transfer 2 ml of 1% Glucose into a 3^(rd) 15 ml tube    -   4) weigh 6 mg of ANS (Ammonium Nickel Sulfate) and transfer it        into 1^(st) 15 ml tube; vortex    -   5) add 110 μl of Glucose Oxidase (Sigma, Cat. No.G-6891) into an        eppendorf tube    -   6) add 110 μl of DAB substrate (Diaminobenzidine HCl, KPL,        Maryland Cat. No.71-00-46) into another eppendorf tube    -   7) Mix in the hood: 5 ml PB+2 ml Glucose        -   +110 μl GO        -   +110 μl DAB        -   +2.8 ml 0.2M PB-   13. Apply detection mixture on the membrane and set up timer. Record    length of incubation in chromogen.-   14. After bands became visible enough wash the membrane 3 times with    0.2M PB.-   15. Shake in PBS overnight at RT.

B. Chemiluminescence Protocol:

-   1. Rinse the membrane twice in TBS-T (Tris-buffered saline    pH7.6+0.1% Tween-20). Tris-buffered saline: 2.42 g Tris base (20 mM)    -   8 g sodium chloride (137 mM)    -   3.8 ml 1M hydrochloric acid-   2. Block overnight in 20 ml of blocking buffer (TBS-T/5% nonfat dry    milk)    -   4° C./shaker:    -   20 ml TBS-T+1 g milk    -   Volumes depend on type of container-   3. Rinse the membrane twice with TBS-T.

4. Incubate the membrane in 5 ml of primary antibody (Affinity ResearchProducts Ltd, United Kingdom) in blocking buffer for 2 hrs at RT/shaker:α-LMP 2 antiserum (mouse) (Cat. No. PW8205) 1:5000 α-LMP 2 antiserum(human) (Cat. No. PW 8345) 1:10000 α-LMP 7 antiserum (Cat. No. PW 8200)1:20000 α-20S proteasome α2 subunit monoclonal 1:1000 antibody (Cat. No.PW 8105)

-   5. Wash the membrane in 20 ml of TBS-T at RT/shaker:    -   Briefly rinse the membrane using two changes of TBS-T then wash        once for 15 minutes and twice for 5 minutes with fresh changes        of the washing buffer at room temperature.-   6. Incubate the membrane in 5 ml of HRP labeled (Horseradish    peroxidase-labeled) secondary antibody (Amersham; Cat# NIF 824 or    NIF 825) 1:1000 dilution in blocking buffer for 1 h at RT/shaker-   7. Wash the membrane as in step 5.-   8. Mix an equal volume of detection solution 1 (Amersham,    Cat#RPN2109) and detection solution 2 (Amersham, Cat#RPN2109) (1    ml+1 ml).-   9. Drain the excess buffer from the washed membrane and put it on a    piece of Saran Wrap, protein side up. Add the detection reagent to    cover the membrane.-   10. Incubate for 1 minute at room temperature without agitation.-   11. Drain off excess of detection reagent and transfer the membrane    to Kodak Digital Science Image Station 440CF protein side down.    Develop and quantify the signal according to the manufacturers'    instructions.

The presence of housekeeping-specific subunits (in either protocol) isdirectly assessed using: α-β1 (Y) subunit monoclonal antibody (Cat. No.PW 8140) 1:1000 α-β2 (Z) subunit monoclonal antibody (Cat. No. PW 8145)1:1000(Affinity Research Products Ltd, United Kingdom).

Example 2 Generation of Predicted MHC I Peptide Cleft Binding PeptidesUsing Algorithmic Modeling

A population of candidate MHC I binding peptides, generated from theamino acid sequence of human carcinoembryonic antigen precursor (CEA)(GENBANK ACCESSION P06731), was produced using an algorithm. Theparticular algorithm is available at<<http://134.2.96.221/scripts/hlaserver.dll/EpPredict.htm>>, asdiscussed above and hereby incorporated by reference in its entirety.Once the algorithm was accessed, the amino acid sequence for CEA wasprovided. Next, parameters for the length of the epitope (decamers) andthe particular MHC allele (H2-Db) of interest were selected. Followingthis, the data were submitted for algorithmic analysis. The resultingdata are shown in Table II. TABLE II Fragments of CEA having PredictedAffinity for H2-Db Seq Id POS 1 2 3 4 5 6 7 8 9 0 Score no+HZ,1/32 547 LQ L S N G N R T L 26 1 369 L Q L S N D N R T L 26 2 191 L Q L S N G N RT L 26 3 53 L L V H N L P Q H L 26 4 371 L S N D N R T L T L 25 5 549 LS N G N R T L T L 24 6 193 L S N G N R T L T L 24 7 299 C Q A H N S D TG L 23 8 100 I I Y P N A S L L I 21 9 578 S A N R S D P V T L 19 10 576S V S A N R S D P V 19 11 504 S I S S N N S K P V 18 12 356 W W V N N QS L P V 18 13 178 W W V N N Q S L P V 18 14 148 S I S S N N S K P V 1815 127 S D L V N E E A T G 18 16 645 I T P N N N G T Y A 17 17 540 S L PV S P R L Q L 17 18 362 S L P V S P R L Q L 17 19 326 F I T S N N S N PV 17 20 250 R S G E N L N L S C 17 21 184 S L P V S P R L Q L 17 22 140V Y P E L P K P S I 17 23 40 S T P F N V A E G K 17 24 29 N P P T T A KL T I 17 25 655 C F V S N L A T G R 16 26 608 G A N L N L S C H S 16 27606 L S G A N L N L S C 16 28 604 S Y L S G A N L N L 16 29 571 C G I QN S V S A N 16 30 496 V S A E L P K P S I 16 31 465 S N I T E K N S G L16 32 453 G N I Q Q H T Q E L 16 33 441 S N P P A Q Y S W L 16 34 393 CG I Q N E L S V D 16 35 242 I S P L N T S Y R S 16 36 91 P G P A Y S G RE I 16 37 43 F N V A E G K E V L 16 38 693 I G V L V G V A L I 15 39 684S A G A T V G I M I 15 40 510 S K P V E D K D A V 15 41 482 S A S G H SR T T V 15 42 428 R P G V N L S L S C 15 43 399 L S V D H S D P V I 1544 372 S N D N R T L T L L 15 45 332 S N P V E D E D A V 15 46 329 S N NS N P V E D E 15 47 307 G L N R T T V T T I 15 48 289 I T V N N S G S YT 15 49 280 S T Q E L F I P N I 15 50 277 F Q Q S T Q E L F I 15 51 222S A R R S D S V I L 15 52 221 V S A R R S D S V I 15 53 154 S K P V E DK D A V 15 54 135 T G Q F R V Y P E L 15 55 49 K E V L L L V H N L 15 5634 A K L T I E S T P F 15 57

The table above arbitrarily cuts off scores below 15. The algorithm canproduce scores of less than 15.

Example 3 Digestion of Peptide Precursors Using Immune and HousekeepingProteasomes to Determine Fragments Produced by Proteolytic Digestion

Peptides were synthesized using a 433A ABI synthesizer. Peptides wereproduced in 0.25 mmole quantities using Fastmoc chemistry. The peptideswere tested for solubility and once solubilized, a 2 mM solution wasprepared and divided into ˜25-30 μL aliquots which were stored at −20°C. for future use. Timed digest reactions, typically consisting of 2 μlof peptide and 4 μl of proteasome, were conducted with t=0 as a controland an incubation of the peptide with water instead of the proteasome asa further control. The reaction was carried out at 37° C. and ended bythe addition of 10% TFA (trifluroacetic acid) on dry ice. The frozensamples were then analyzed by MALDI-TOF mass spectroscopy (MS) asdescribed in Example 4, below.

An optional desalting step can be performed on the digests prior to MSanalysis using the ZIP-TIP method (Millipore, Boston, Mass.). The ZIPTIP is a specially designed pipette tip which contains a bed ofspherical silica resin. The sample is bound to the tip, which ispre-equilibrated with 0.1% TFA, and then eluted with 50% Acetonitrile0.1% TFA elution buffer.

Example 4 Identification and Quantitation of Relevant ProteolyticFragments by HPLC and Mass Spectrometry

A. Identifying Sequences of Therapeutic Interest

The amino acid sequence of a protein of interest is entered into acomputer, and the algorithm of Rammensee, et al., is used to generate 9-or 10-amino-acid-long sequences predicted to bind a particular HLAreceptor. The algorithm also ranks these predicted epitopes according tohow well they match the binding motif.

Synthetic peptides containing the sequence of the identified potentialepitopes are then constructed to encompass the epitope candidatesequence and at least 3-5 residues proximal to its termini. The residuesadded to the ends of a particular epitope candidate are to ensure thatthe proteasome complex encounters a processing environment similar tothat found within the cell, hence increasing the likelihood that itperforms its proteolytic functions normally. Additional residuesnormally found proximal to the ends of the epitope candidate may beadded if necessary to help increase the solubility of the peptides.

Peptides are synthesized on an Applied Biosystems 433A PeptideSynthesizer (Applied Biosystems, Norwalk, Conn.) using standard Fmocsolid phase synthesis methodologies. The synthesizer is equipped with aconductivity feedback monitoring system which allows for increasedreaction times for sequences that contain stretches of difficult todeprotect and difficult to couple residues. After synthesis, thepeptides are cleaved from their support with trifluoroacetic acid in thepresence of appropriate scavengers, precipitated with ether and thenlyophilized.

The crude peptides are then dissolved in a suitable solvent at 0.5mg/ml. Five microliters (5 μl) of this solution is then analyzed on aShimadzu analytical reverse phase HPLC system (Shimadzu ScientificInstruments, Columbia, Md.) using a 0.1% TFA water-acetonitrilegradient. Typically, a C-18 silica column (Machery-Nagel # 720051.40,(Machery-Nagel GmbH, Germany)) is used for hydrophillic and a phenylsilica column (Vydac # 219TP5415 (The Separations Group, Inc., Hesperia,Calif.)) is used for hydrophobic peptides. The gradients used vary from0-40% acetonitrile for hydrophillic to 30-70% acetonitrile forhydrophobic peptides. The peptides are subsequently purified on a VarianProstar HPLC system (Varian, Inc., Palo Alto, Calif.) using similargradients and semi-preparative versions of the above-mentioned columns(Machery Nagel # 715802, and Vydac 219TP510). The major HPLC fractionsfrom the first preparative injection of the peptide are analyzed using aMALDI-TOF mass spectrometer to identify the desired component. Thecorresponding peaks from subsequent injections are collected, pooled andlyophilized, and a sample is taken to verify retention time andchromatographic purity by analytical HPLC using the system describedabove. These purified peptides are then ready for digestion by theproteasome preparation.

B. Proteasome Assay

Immune or housekeeping proteasome complexes are isolated by the methodof Levy, (Morel, S., et al., Immunity 12:107-117 (2000), and thereferences cited therein, which are all incorporated by reference intheir entireties) described above. The purified peptide is dissolved inan appropriate buffer to a concentration of about 1 to 2 mM and added toapproximately 2 volumes of the proteasome preparation. The buffer chosenmust solvate the peptide without interfering with the digestion process.An additional digest is prepared using the positive control peptidedescribed above to verify proper functioning of the proteasomepreparation used. These are incubated at 37° C. for periods of up to 120minutes and then the digestion is stopped by the addition of dilutetrifluoroacetic acid; the samples are analyzed immediately by massspectrometry, or they are frozen on dry ice until analysis. The digestreaction can also be halted by putting samples on ice for immediateanalysis by mass spectrometry.

C. MALDI-TOF Mass Spectrometric Analysis of the Digest

Approximately 0.5 μl of each digest was mixed with an equal volume ofthe matrix solution (10 mg/ml dihydroxybenzoic acid in 70% EtOH, pH 2-3)directly on the sample slide and allowed to air dry at about 40° C. Thesamples were then analyzed on a Lasermat™ MALDI-TOF mass spectrometer(Thermo Bioanalysis, Santa Fe, N. Mex.) that was calibrated withsuitable molecular weight standards.

The computer programs (either “Peptide” software, (Lighthouse Data), or“Dynamo” (ThermoBioanalysis Ltd., U.K.)) developed for the proteasomeassay generates the sequence and molecular weight of all the possiblefragments that satisfy both requirements of having the correctC-terminus of any predicted epitope, and of containing the full lengthof that epitope or longer.

When the MALDI-TOF results showed that a particular molecular weight wasrepresented in a digestion mixture, the corresponding peptide wassynthesized, purified, identified by MALDI-TOF and then subjected toreverse phase analytical HPLC to establish a standard retention time andan approximate mass to peak area ratio. These procedures are directlyanalogous to those described above. A replicate proteasome digest wasthen diluted in an appropriate solvent and analyzed using the sameanalytical HPLC method. When the digest gives a peak in good yield thathas the same retention time as that of the standard, it is almostcertain that it is due to the presence of that sequence in the digest.When there is any ambiguity due to the possible generation of otherfragments that would give rise to the same or similar mass spectrometryresults, the suspect component can be collected and set aside forsequencing to confirm identity. The analytical HPLC also importantlyprovides relatively accurate quantitation of the peptide product in thedigest, which allows determination of whether a given peptide is a minoror a major product of the digest, which indicates whether the epitope isefficiently produced by the proteasome. Using the above method,housekeeping epitopes were identified. FIG. 2 shows the results of aflow cytometry assay to verify HLA binding by these epitopes. This assayis discussed in Example 5.

Example 5 Determine the MHC Binding Ability of Selected Peptides

Binding of a candidate epitope to HLA-A2.1 was assayed according to themethod of Stauss et al., (Proc Natl Acad Sci USA 89(17):7871-5 (1992)).T2 cells, which express empty or unstable MHC molecules on theirsurface, were washed twice and suspended at 5×10⁶ cells/ml in serum-freecomplete Iscove's modified Dulbecco's medium (IMDM). β₂ microglobulin(Sigma, St. Louis, Mo.) was added at 5 μg/ml and the cells distributedto a 96-well U-bottom plate at 5×10⁵ cells/well. Peptides were added at100, 10, 1 and 0.1 μg/ml. The plate was rocked gently for 2 minutes andthen incubated for 4 hours in a 5% CO₂ incubator at 37° C. After theunbound peptide was removed by washing twice with IMDM, a saturatingamount of monoclonal antibody W6/32 (Sigma) was added. After incubationfor 30 minutes at 4° C., cells were washed with PBS supplemented with 1%heat-inactivated FCS, 0.1%(w:v) sodium azide, pH 7.4-7.6 (stainingbuffer), and incubated with fluorescein isothiocyanate (FITC)-conjugatedgoat F(ab′) antimouse-IgG (Sigma) for 30 min at 4° C. and washed fourtimes as before. The cells were resuspended in staining buffer and fixedby adding a quarter volume of 2% paraformaldehyde. The analysis ofsurface HLA-A2.1 molecules stabilized by peptide binding was performedby flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.).

The results of the experiment are shown in FIG. 3. Using the methoddiscussed above, a candidate tyrosinase housekeeping epitope identifiedby proteasomal digestion, (tyrosinase 207-216, FLPWHRLFLL SEQ ID NO: 60)was found to bind HLA-A2.1 to a similar extent as the known A2.1 binderFLPSDYFPSV (SEQ ID NO: 61)(positive control). HLA-B44 binding peptideAEMGKYSFY (SEQ ID NO: 62) used as a negative control. The fluorescenceobtained from the negative control was similar to the signal obtainedwhen no peptide was used in the assay. Positive and negative controlpeptides were chosen from Table 18.3.1 in Current Protocols inImmunology p. 18.3.2, John Wiley and Sons, New York, 1998.

1. A method for selecting and/or producing a T cell epitope, comprisingcontacting a peptide or polypeptide to a proteasome to permit processingof the peptide or polypeptide by the proteasome to determine thec-terminus of said T cell epitope.
 2. The method of claim 1, whereinsaid proteasome is an immune proteasome.
 3. The method of claim 1,wherein said proteasome is a housekeeping proteasome.
 4. The method ofclaim 1, wherein said proteasome is a proteasome exposed tointerferon-gamma.
 5. The method of claim 1, wherein said peptide orpolypeptide is synthetic.
 6. The method of claim 1, wherein said peptideor polypeptide is a fragment of an antigen.
 7. The method of claim 1,wherein said peptide or polypeptide is an epitope cluster.
 8. The methodof claim 1, further comprising identifying additional characteristics ofsaid T cell epitope.
 9. The method of claim 8, wherein saidcharacteristics comprises at least one characteristic selected from thegroup consisting of MHC binding affinity, identity of a c-terminus,N-terminus, and proteolytic cleavage sites.
 10. A T cell epitopeobtainable by a method selected from the group consisting of the methodof claim 1, 2, 3, 4, 5, 6, 7, 8 and
 9. 11. A nucleic acid encoding atleast one T-cell epitope according to claim
 10. 12. The nucleic acid ofclaim 11, comprising at least two sequences, each encoding a T cellepitope, wherein said at least two sequences are separated by at leastone proteolytic cleavage site.
 13. A vector comprising a nucleic acidselected from the group consisting of the nucleic acid of claim 11 andclaim
 12. 14. A composition comprising a vector of claim
 13. 15. Acomposition comprising a T cell epitope of claim
 10. 16. A polypeptideobtainable by expressing a nucleic acid of claim 11 or claim
 12. 17. Acomposition comprising the polypeptide of claim
 16. 18. A compositioncomprising an adjuvant and a composition selected from the groupconsisting of the composition of claim 14, 15 and 17 the polypeptide ofclaim
 16. 19. A method for selecting and/or producing a T cell epitope,comprising subjecting a precursor peptide or polypeptide to the actionof a 20S proteasome, in particular an IFN-.gamma. or otherimmune-related proteasome or a functional equivalent thereof todetermine the location of the c-terminus of said T cell epitope.
 20. Amethod for selecting and/or producing according to claim 1 wherein saidprecursor peptide or polypeptide is tested for other T cell epitopecharacteristics.
 21. A method according to claim 20, wherein saidfurther testing comprises testing for the right anchor residues, theproper cleavage sites, the proper pathway signals, and/or the stabilityof the MHC-T cell epitope complex.
 22. A T cell epitope obtainable by amethod according to any one of claims 19-21.
 23. A nucleic acid encodingat least one T cell epitope according to claim
 22. 24. A nucleic acidaccording to claim 23 comprising at least two sequences encoding a Tcell epitope, separated by at least one proteolytic cleavage site.
 25. Agene delivery vehicle comprising a nucleic acid according to claim 22 or23.
 26. A pharmaceutical composition comprising a gene delivery vehicleaccording to claim
 7. 27. A pharmaceutical composition comprising atleast one epitope according to claim
 22. 28. A proteinaceous moleculeobtainable by expression of a nucleic acid according to claim 23 or 24.29. A pharmaceutical composition comprising a proteinaceous moleculeaccording to claim
 28. 30. A pharmaceutical composition according toclaim 26, 27 or 29 further comprising an adjuvant.
 31. A pharmaceuticalcomposition according to claim 26, 27, 29 or 30 comprising an antigenpresenting moiety.
 32. A pharmaceutical composition according to claim31 wherein said moiety comprises a major histocompatibility molecule.33. A pharmaceutical composition according to claim 32 wherein saidmajor histocompatibility molecule is present on an antigen presentingcell, such as a dendritic cell.
 34. A method of epitope discoverycomprising the step of selecting an epitope from a population of peptidefragments of an antigen associated with a target cell, wherein thefragments have a known or predicted affinity for a majorhistocompatibility complex class I receptor peptide binding cleft,wherein the epitope selected corresponds to a proteasome cleavageproduct of the target cell.